Transcriptionally Engineered Addressable RNA Solvent droplets as generalised synthetic organelle – SYNORG

In general, the efficiency of metabolic pathways is limited by short lifetime, toxicity and diffusion of intermediates, and by other competing pathways. These caveats were met in nature by the evolution of isolated microenvironments as organelles. With the advent of synthetic biology, intended to expand on natural metabolic capabilities, the implementation of exogenous synthetic pathways is limited by the lack of such compartments in popular workhorse for bioproduction as E. coli. This bottleneck hampers synthetic biology contribution to meet with sustainable development challenges by providing cost- and carbon-effective potential solutions. Here, we exploit RNA-driven liquid-liquid phase separation (LLPS) to design modular Transcriptionally Engineered Addressable RNA Solvent droplets (TEARS). We previously demonstrated that rCAG long repeats-based TEARS are capable of controlling metabolic pathways by recruiting multiple proteins via RNA aptamer-adaptors while causing minimal cell growth burden. In this project, our main objective is to decipher the mechanisms underlying formation and properties of LLPS-based RNA intracellular compartmentalisation in order to achieve diversified and modular synthetic organelles in bacteria for biotechnological applications. To this end, we will couple iterative biocomputation- and physics-based model predictions with systemic Design-Build-Test-Learn experimental cycles to infer RNA-driven LLPS sequence-dependent rules, characterise their bio-physical and -chemical properties of diversified TEARS and harness them for recruiting RNA and protein clients for fundamental understanding and biotechnological applications.